Applicant's Abstract Release of calcium ions from the sarcoplasmic reticulum via type 2 ryanodine receptors (RyR2) is an integral step in the cascade of events leading to cardiac muscle contraction. Studies have shown that this process is compromised in heart of diabetic rats (Yu and McNeill 1991; Yu et al. 1994). We recently found that the decrease in activity of RyR2 stems from a dysfunction of this protein rather than a decrease its expression (Bidasee et al manuscript #1). To date, the molecular basis for the dysfunction of RyR2 is not known. Our working hypothesis is "diabetes alters the integrity of the calcium efflux pathway on RyR2." In this project we want to characterize changes in RyR2 induced by diabetes and to determine the effects of these changes on the regulation of RyR2 by endogenous modulators. At the same time, we want to investigate whether the beneficial effects of insulin and verapamil treatments include reversal of diabetes-induced changes to RyR2 protein. Our specific aims are: (1) to identify and characterize molecular changes to RyR2 protein induced by diabetes, (2) to ascertain whether these changes alter the sensitivity of RyR2 to endogenous modulators like Ca2+, pH etc., (3) to determine whether changes to RyR2 induced by long-term diabetes can be reversed with insulin treatment, (4) to determine whether verapamil treatment can protect and/or reverse diabetes-induced changes to RyR2, and (5) to establish if verapamil and insulin co-treatments have additive effects on reversing changes to RyR2 induced by diabetes. Accomplishment of these aims will contribute significantly to understanding the molecular basis for the decrease activity of RyR2 protein (and possibly other proteins) in diabetes and this could lead to newer insights into therapeutic strategies for alleviating diabetes-induced dysfunction of the heart.